Structural Biology of RNA Splicing

描述(由申请人提供)： RNA剪接的项目摘要/摘要结构生物学。RNA剪接是真核生物赖以生存的基本生物学机制。它包括从前信使RNA中切除非编码内含子，并连接相邻的外显子以产生成熟的mRNA供核糖体翻译。剪接体内含子约占人类基因组的30%，关于剪接体催化其切割机制的确切结构细节知之甚少。第二组内含子被认为是剪接体内含子的祖先，因此我们使用第二组内含子作为一个易于处理的模型系统，以获得对真核细胞剪接的详细分子见解。此前，等电点的工作是确定第二类内含子的第一个晶体结构。这一结构揭示了内含子通过双金属离子机制拼接。然而，这个结构是一个水解性的线性II组内含子，它缺少一个称为结构域6的重要区域。这个关键的结构域包含膨胀的腺苷，负责形成被称为套索的分支RNA。套索由膨大的腺苷的核糖和内含子的5‘端之间不寻常的2’-5‘磷酸二酯键组成。剪接体内含子中类似的基序称为分支点。分支位置的突变导致套索形成缺陷，这是几种人类疾病的原因。套索形成是剪接体正确选择剪接位点所必需的，被认为是真核剪接的特征标志。我们的长期目标是通过研究套索形成第二组内含子来了解真核剪接机制。我们将使用生化和结构方法来：1)确定套索形成的结构基础。2)分析剪接途径中的催化中间产物，以确定与剪接过程相关的构象变化。这有望与真核生物中剪接体催化的RNA剪接有直接的相似之处。

DESCRIPTION (provided by applicant): PROJECT SUMMARY/ABSTRACT Structural biology of RNA splicing. RNA splicing is a fundamental biological mechanism essential for the survival of every eukaryotic organism. It involves the excision of non-coding introns from pre-messenger RNAs and ligation of the adjacent exons to generate mature mRNA for translation by the ribosome. Spliceosomal introns comprise ~30% of the human genome and there is relatively little known about the precise structural details of their mechanism of excision catalyzed by the spliceosome. Group II introns are considered to be the ancestors of spliceosomal introns, therefore we are using the group II intron as a tractable model system to gain detailed molecular insight into eukaryotic splicing. Previously, the PI worked to determine the first crystal structure of a group II intron. This structure revealed that introns splice via a two-metal ion mechanism. However, this structure was of a hydrolytic, linear group II intron that was missing an important region called domain 6. This crucial domain contains the bulged adenosine responsible for forming the branched RNA known as a lariat. The lariat consists of an unusual 2'-5' phosphodiester bond between the ribose sugar of the bulged adenosine and the 5' end of the intron. The analogous motif in spliceosomal introns is called the branch site. Mutations in the branch site lead to defects in lariat formation that are the cause of several human diseases. Lariat formation is essential for proper splice site selection by the spliceosome and is considered to be a characteristic hallmark of eukaryotic splicing. Our long-term goal is to gain knowledge of eukaryotic splicing mechanisms by studying a lariat-forming group II intron. We will use biochemical and structural approaches to: 1) Determine the structural basis for lariat formation. 2) Analyze catalytic intermediates in the splicing pathway to determine the conformational changes that are associated with the progression of splicing. This is expected to have direct parallels with RNA splicing catalyzed by the spliceosome in eukaryotes.